Supplementary Information
CHARACTERIZATION OF A HEXAMERIC EXO-ACTING GH51 α-LARABINOFURANOSIDASE FROM THE MESOPHILIC Bacillus subtilis
Zaira B. Hoffmam, Leandro C. Oliveira, Junio Cota, Thabata M. Alvarez, José A.
Diogo, Mario de Oliveira Neto, Ana Paula S.Citadini, Vitor B. P. Leite, Fabio M.
Squina, Mario T. Murakami and Roberto Ruller
SDS-PAGE of purification steps for recombinant AbfA
Figure S1: MW indicates the standard molecular weight markers. The single band represents the AbfA
(59,5 kDa) purified (line 1) from nickel-affinity chromatography and (line 2) after polishing step by sizeexclusion chromatography.
Activity against distinct polysaccharides
Table S1: AbfA activity over different polysaccharides.
Polysaccharide
Glucose equivalent
(µmol/mL)
Debranched Arabinan
1.66±0,05
Linear arabinan
0.59±0,02
Sugar beet
0.40±0,05
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Small Angle X-Ray Scattering (SAXS) and mass estimation
SAXS is an experimental technique extensively employed to characterize the
structure of protein in terms of the shape and averaged particle distribution. Generally,
SAXS is analyzed by the scattering profile generated (Intensity (I) versus the momentum
transfer (q)) that allow the construction of envelopes (shape representation) and the
calculation of the pair-distance distribution function (P(r)) where possible conformational
changes can be analyzed [1]. The combination of SAXS and computational simulations
improve the accuracy of the method and to promote a good kinetic evaluation of the
protein in solution. An important region of the scattering profile is defined by the low “q”
region, also known as Guinier region, where is possible to extrapolate the value of
intensity to q~0 and to estimate the Radius of Guinier (Rguinier). The initial part of the
curve also is important to estimate the mass that must be distributed into a given volume.
The Figure 2 shows the theoretical scattering profiles to conformations with different
mass (composed by a monomer or monomers) and radius of gyration, illustrating the
importance of the correct mass and shape for good fitting with the experimental data.
There, it is presented the poor fitting using the monomeric form, an monomeric form
extended, and an compact dimer, trimer, tetramer and pentamer. For all these cases
evaluated the fitting is just not satisfactory. The values of Rg, Rguinier and χ2 to Figure 2
are presented in Table S2.
SAXS Analysis through ATSAS package
SAXS data was also evaluated in this work by old-fashioned methods, i.e.
employing the construction of envelops and by bringing many monomeric structures
2
together. On the SAXS evaluation the radius of gyration (Rg) was approximated using
two independent procedures, by Guinier equation and by indirect Fourier transform
method using GNOM program [2]. The distance distribution function P(r) was evaluated
by GNOM and the maximum diameter (Dmax) obtained calculating the most distant
atoms in the protein. Additionally, the Rigid Body Model (RBM) simulations using
SASREF [3] were performed to trimeric, tetrameric, pentameric, hexameric and
heptameric constructions without symmetry imposition. The best conformation provided
by RBM calculation was hexameric, corroborating to results obtained by the web server
SAXSMoW (see Figure S3).
SAXS envelopes were obtained using dummy atoms model (DAM) where the
shape was calculated from the experimental SAXS data using the procedures
implemented in GASBOR [4], DAMMIN [5] and DAMMIF [6] included in ATSAS
package v2.4 [7]. Several runs of ab initio shape determination were performed until
reaching a consistent results, judged by the structural similarity of the output models
yielding nearly identical scattering patterns and fitting statistics in a stable and selfconsistent process.
As presented in the manuscript, lower values of χ2 means a better agreement
between the theoretical and experimental scattering curves. The best DAM generated by
GASBOR and recalculated by CRYSOL presented a χ2 =5.6 with Rg=45.9 Å and the
structure docked in with the 6 monomeric repetitions in the SAXS envelop provided a χ2
=5.3. DAMMIN calculations generate an envelope with χ2 =4.7 and Rg=47.1 Å and
DAMMIF a χ2 =8.9 and Rg=47.2 Å. The values obtained can be found on table S1. As it
is possible to verify, the envelopes generated by the programs are less accurate than the
results obtained using all-atoms SBM.
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Monomeric models were obtained through sequence alignments based on the
primary sequence. The crystal structure with the highest sequence identity 72.1% (PDB
code: 1PZ3) was employed to reconstruct the arrangement into SAXS envelope. The
arrangements obtained by DAM and RBM were compared to the hexameric crystal
structure with the highest sequence identity 63.6% (PDB code: 2C7F) that presented a χ2
=5.5 and Rg=45.9 Å.
Table S2: Gyration radius and χ2 of the conformations presented in figure 2.
Construction
Rg (Å)
Rguinier (Å)
χ2
Chain A
23.3
50.6
25.8
Chain A (ext)
42.4
47.5
25.1
Chain AB
35.9
50.6
24.4
Chain ABC
42.1
47.6
22.4
Chain ABCD
44.9
47.5
16.9
Chain ABCDE
45.5
47.3
12.8
Radius of gyration (Rg) and Radius of Guinier calculated using the program CRYSOL.
4
Table S3: Structural parameters obtained from SAXS analysis for AbfA
Parameter
Exp
DAMMIN DAMMIF GASBOR SBM
AbfA*
Dmax(Å)
140 ±5
134.6
149.5
133.3
149.0 132.9
Rg (Å)
46.6±0.1
47.1
47.2
45.9
46.4
45.9
4.7
8.9
5.6
4.5
5.5
(GNOM)
47±1.6 (Guinier)
χ2
MWSAXS
352.8kDa**
356.1kDa
(SAXSmow)
* Based on the atomic coordinates generated by molecular modeling using the PDB 2C7F as template
** Calculated using the AbfA amino-acid sequence
5
Figures
Figure S2: Experimental scattering profile compared to theoretical curves for distinct tertiary conformations
(chain A and chain A extended by high temperature simulations) and quaternary arrangements (chains A
and B; chains A, B and C; chains A, B, C and D; and chains A, B, C, D and E). Neither of the theoretical
curves provided a good fitting, since the mass distribution of the conformations and shape are not correct.
6
Figure S3: Screen Capture of SAXSMoW calculation. The web server estimated the molecular weight
from the experimental SAXS data as 356 kDa that corresponds to a hexameric quaternary arrangement.
7
Figure S4: The atomic coordinates of AbfA (blue cartoons) fitted into the low-resolution SAXS envelope
calculated using the program GASBOR. The image was generated using PyMol [8]
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[8] The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.
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